Isoflavone metabolites

ABSTRACT

There are disclosed compounds of formulae (I) or (II) in which A is selected from the group consisting of (1), (2), (3) and (4); OH, and one of R 1  and R 2  is selected from H, OH and OCH 3 , and the other of R 1  and R 2  is selected from OH and OCH 3 ; one of R3 and R4 is selected from H, OH and OCH3, and the other of R3 and R4 is selected from OH and OCH3; provided that at least one of the pairs R 1 , R 2  and R 3 , R 4  are both OH; R 5  is selected from OH and OCH 3 ; and    
     denotes a single or double bond; and pharmaceutically acceptable salts and prodrugs thereof. The compounds of the invention are useful for the treatment of hormone-dependent conditions and cancers.

FIELD OF THE INVENTION

This invention relates to certain isoflavonoid compounds, compositionscontaining the same, and therapeutic uses of those compounds.

BACKGROUND OF THE INVENTION

In recent years there has been increasing attention on phytoestrogensparticularly isoflavonoids. Isoflavonoids or isoflavones (as they arealso known) are a class of phytoestrogens which are found in plants andwhich are based on a diphenolic ring structure. Due to their structure,it has been documented that they are able to bind to oestrogen receptorson animals including humans. A small subgroup of isoflavones are knownto display oestrogenic activity, as well as anti-carcinogenic,antifungal, antiproliferative properties and anti-oxidative effects.These oestrogenic isoflavones (genistein, biochanin, daidzein, glyciteinand formononetin) are predominantly found in plants which are members ofthe Leguminosae family.

Most legumes have been found to contain at least one or more of theseoestrogenic isoflavones, with the richest sources being soya beans,lentils, clover, chick peas, alfalfa and other beans. Most human dietscontain low to moderate levels of oestrogenic isoflavones. In typicaldiets in developed Western countries, the dietary intake of theoestrogenic isoflavones is low and often negligible, as legumes are notrelied upon strongly as a source of protein, being instead replaced byanimal products.

However, the dietary intake of oestrogenic isoflavones from traditionaldiets of Eastern and developing countries such as India, China and SouthAmerica is moderate to high, given the fairly high dietary intake ofbeans including soya beans, kidney beans, lima beans, broad beans,butler beans, chick peas and lentils. The presence of such dietarylevels of oestrogenic isoflavones is confirmed by detection of theamounts of the isoflavones daidzein, genistein, glycitein, formononetinand biochanin and their metabolites in human urine. People with highlegume intake in their diets excrete substantially higher amounts ofisoflavone metabolites in their urine than people with largelyomnivorous or low-legume diets.

After ingestion, isoflavones undergo varying degrees of metabolismwithin the digestive system. The naturally occurring, water solubleglycosidic form of isoflavone undergoes hydrolysis to the aglycone formin the gut, while biochanin and formononetin are demethylated bybacterial fermentation to genistein and daidzein respectively. Itappears that the majority of the aglycone isoflavones then undergofermentation by intestinal bacteria to produce end products includingequol, dehydroequol, O-desmethylangolensin (ODMA), dihydrodaidzein,tetra-hydrodaidzein and dihydrogenistein. The isoflavones, theirmetabolites and derivatives circulate around the body and are mainlyexcreted in the urine, in which they can then be detected.

As stated above, given the presence of high levels of isoflavones inlegumes, particularly soya beans, and the knowledge that the isoflavonesare fermented or metabolised by intestinal or bowel bacteria to produceisoflavone metabolites, research has been conducted into microbialfermentations of soybeans and has demonstrated production of metabolitesincluding 6,7,4′-trihydroxyisoflavone (hereinafter called Factor 2) andother polyhydroxylated isoflavonoids.

Traditional Asian food products such as tempeh, tofu, miso etc are foodsproduced from soybeans by fermentation mainly by fungi of the genusRhizopux. It has been shown that several bacteria species may also beinvolved in tempeh production. For traditional tempeh fermentation, thesoybeans are cooked, dehulled and soaked overnight. A spontaneousbacterial acidification occurs during this phase. In industrial tempehfermentation processes, the cooked soybeans are acidified with lacticacid. After the soaking process, the soybeans are cooked again andincubated with microbial inocula for 2 days.

In unfermented soybeans, the isoflavones genistein, daidzein andglycitein predominantly occur as isoflavone glucosides andacylglucosides. It has been shown that during tempeh fermentation, theisoflavone aglycones are liberated from the conjugates and accumulate inthe tempeh product. Further findings have shown that during fermentationthe isoflavone 6,7,4′-trihydroxyisoflavone (termed “Factor 2” by Gyorgyet al. in Nature (1964) 203, 870-872), also accumulates.

It was previously thought that the fungi of the genus Rhizopus wereresponsible for the formation of Factor 2 from either daidzein orglycitein. However, subsequent studies on the metabolism of daidzein andglycitein by Klus et al., 1993 showed that isolates of Brevibacteriumepidermidis and Micrococcus luteus, which were isolated from Indonesiantempeh samples, readily transform glycitein, forming Factor 2. A thirdtempeh-derived bacterium. Microbacterium arborescens, metabolizeddaidzein, producing both Factor 2 and glycitein. More recently, Klus, K.and Barz, W. Arch. Microbiol. 164:428-434, (1995) investigated fiveother bacterial isolates, which were isolated from tempeh samplescontaining Factor 2 and were classified as Micrococcus or Arthrobacterstrains, for their ability to metabolize daidzein and glycitein byhydroxylation or O-demethylation reactions. Their results show that anumber of polyhydroxylated isoflavones were formed, hydroxylated atthree or four of positions 6,7,8,3′ and 4′. Of these Factor 2 was themajor product produced by most of the microbial strains. The bacterialstrains only hydroxylated but did not degrade the substrates namelydaidzein or glycitein. The compounds of the present invention were notidentified by Klus and Barz, however,

Various polyhydroxylated isoflavones known in the prior art are known toexhibit anti-inflammatory and anti-allergenic activity and to expressanticarcinogenic properties due to inhibition of protein tyrosinekinases, which play a key role in cellular pathways in tumour cellgrowth. In in vitro tests, these isoflavones also inhibit the growth ofhuman leukemia (Makishima et al., 1991) and human breast cancer cells(Hirano et al, 1989; Peterson and Barnes, 1991). In essence, thepolyhydroxylated isoflavones occurring as dietary factors in fermentedsoybean products are putative causes of the lower incidence ofcancer-related diseases in Asian populations, and have been used in thetreatment of a variety of cancers including breast cancer, ovariancancer, large bowel cancer; and prostatic cancer.

Other therapeutic uses of the oestrogenic isoflavones which have beendisclosed include their use as therapeutics for menopausal symptoms andosteoporosis (WO 98/50026, European patent application 0135172, U.S.Pat. No. 5,498,631 in the name of Gorbach el al); pre-menstrualsymptoms; Reynauds Syndrome; rheumatic diseases; Buergers Disease;coronary artery spasm; migraine headaches; benign prostatic hypertrophyand hypertension.

As stated above, isoflavonoids are natural plant compounds which possessantitumorigenic properties. Of all oestrogenic isoflavones of whichdaidzein, genistein, formononetin and biochanin-A are the most wellknown, it has been shown that individually, genistein is the most potentinhibitor (IC50=25-33 μM) of the proliferation of MCF-7 cells induced bya number of environmental chemicals such as1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane,5-octylphenol and 4-nonylphenol as demonstrated recently by Verma S Pand Goldin B R (Nutrition & Cancer 30(3):232-9,1998).

The same authors also noted that a mixture of isoflavones was the mostpotent inhibitor against the induced proliferation. However, as in thecase of other research workers they found that genistein, biochanin A,equol and to some extent daidzein at <10 μM can enhance the growth ofMCF-7 cells.

There is therefore a need for novel isoflavonoids which can inhibit theproliferation of cancer cells but which do not enhance their growth atlow concentrations, and which exhibit other therapeutic properties.

OBJECTS OF THE INVENTION

It is therefore an object of the present invention to provide novelisoflavonoid compounds.

It is another object of the present invention to provide compositionsincluding food and drink compositions containing novel isoflavonoidcompounds.

It is a further object of the present invention to utilise novelisoflavonoid compounds in treating hormone dependent conditions andother diseases and disorders.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”or the term “includes” or variations thereof, will be understood toimply the inclusion of a staled element or integer or group of elementsor integers but not the exclusion of any other element or integer orgroup of elements or integers.

According to a first aspect of the present invention there is provided acompound of formula I or formula II

in which

A is selected from the group consisting of

one of R₁ and R₂ is selected from H, OH and OCH₃, and the other of R₁and R₂ is selected From OH and OCH₃;one of R₃ and R₄ is selected from H, OH and OCH₃, and the other of R₃and R₄ is selected from OH and OCH₃;provided that at least one of the pairs R₁, R₂ and R₃, R₄ are both OH;

R₅ is selected from OH and OCH₃; and

denotes a single or double bond,or a pharmaceutically acceptable salt or prodrug thereof.

In one form, the invention relates to compounds of formula (I) or (II)as defined hereinabove, wherein

one of R₁ and R₂ is selected from H and OH, and the other of R₁ and R₂is OH;one of R₃ and R₄ is selected from H and OH, and the other of R₃ and R₄is OH;provided that at least one of the pairs R₁, R₂ and R₃, R₄ are both OH;

R₅ is OH; and

denotes a single or double bond.

In another form, the invention relates to compounds of the formula (IA)or (IIA)

wherein A is as defined hereinabove

R₂ is H, and R₁ is selected from OH and OCH₃; R₃ and R₄ are each OH; R₅is selected from OH and OCH₃; and

denotes a single or double bond.

In a further form, the invention relates to compounds of the formula(IB) or (IIB)

wherein A is as defined hereinabove

R₁ and R₂ are each OH; R₄ is H, and R₃ is selected from OH and OCH₃; R₅is selected from OH and OCH₃; and

denotes a single or double bond.

Examples of preferred compounds of the invention are:

(i) 4′,6,7-trihydroxydihydroisoflavone having the structure (III):

(hereinafter referred to as Compound B);

5-hydroxy-O-demethylangolesin (5-hydroxy-O-Dma)[1-(2,4,5-trihydroxyphenyl)-2-(4-hydroxyphenyl)-propan-1-one] having thestructure (IV):

(hereinafter referred to as Compound A);

3′-hydroxy-O-demethylangolesin (3′-hydroxy-O-Dma)[1-(2,4,dihydroxyphenyl)-2-(3,4-dihydroxyphenyl)-propan-1-one] havingthe structure (V):

3′-hydroxy-O-demethyldehydroangolesin (3-hydroxydehydro-O-Dma)[1-(2,4-dihydroxyphenyl)-2-(3,4-dihydroxyphenyl)-prop-2-en-1-one] havingthe structure (VI):

3′-hydroxy-dihydrodaidzein having the structure (VII):

5-hydroxy-2-dehydro-O-Dma[1-(2,4,5-trihydroxyphenyl)-2-(4-hydroxyphenyl)-prop-2-en-1-one] havingthe structure (VIII):

or pharmaceutically acceptable salts or prodrugs thereof.

A third aspect of the present invention provides a compositioncomprising one or more compounds of the formulae I or II as previouslydefined, in association with one or more pharmaceutically acceptablecarriers, adjuvants, diluents and/or excipients.

Typically, one or more of the compounds of structures (III) to (VIII)may be used in a composition of the third aspect of the presentinvention.

A fourth aspect of the present invention is a food or drink composition,which contains one or more compounds of the formulae I or II.

Typically, the food or drink composition contains one or more of thecompounds of structures (III) to (VIII).

According to a fifth aspect of the present invention there is provided amethod for the treatment, prophylaxis, amelioration, defence against,and/or prevention of menopausal syndrome including depression, anxiety,hot flushes, night sweats, mood swings, and headache; osteoporosis;rheumatic diseases; atherosclerosis; premenstrual syndrome, includingfluid retention, cyclical mastalgia, and dysmenorrhoea; coronary arteryspasm; vascular diseases including Reynauds Syndrome; Buergers Disease;migraine headaches; hypertension; benign prostatic hypertrophy; allforms of cancer including breast cancer, endometrial cancer, prostaticcancer, uterine cancer, ovarian cancer, testicular cancer, large bowelcancer; Alzheimers disease; inflammatory diseases including Crohnsdisease, inflammatory bowel disease, ulcerative colitis; baldnessincluding male pattern baldness; psoriasis; acne; and diseasesassociated with oxidant stress including myocardial infarction, sunlightinduced skin damage, arthritis, or cataracts, which method comprisesadministering to a subject a therapeutically effective amount of one ormore compounds of the formulae I or II as previously defined, eitheralone or in association with one or more pharmaceutically acceptablecarriers, diluents, adjuvants and/or excipients.

According to a related sixth aspect of the present invention there isprovided a method for the treatment, prophylaxis, amelioration, defenceagainst, and/or prevention of hormone-dependent conditions includinghormone dependent cancers such as breast cancer, hormone dependentcardiovascular disorder and hormone dependent menopausal disorderscomprising administering to a subject a therapeutically effective amountof one or more compounds of the formulae I or II as previously defined,either alone or in association with one or more pharmaceuticallyacceptable carriers, diluents, adjuvants and/or excipients.

Typically, one or more of the compounds of structures (III) to (VIII)may be used in the method of treatment, prophylaxis, amelioration,defence against, and/or prevention of any one or more of the diseases ofthe fifth or sixth aspects of the invention.

A seventh aspect of the present invention is the use of one or morecompounds of the formulae I or II for the manufacture of a medicamentfor the treatment, amelioration, defence against, prophylaxis and/orprevention of one or more of the diseases set out in the fifth or sixthaspects of the invention above.

It is typical that one or more of the compounds of structures (III) to(VIII) are employed in the seventh aspect of the present invention.

A related eighth aspect of the present invention is use of one or morecompounds of the formulae I or II in the treatment, amelioration,defence against, prophylaxis and/or prevention of one or more of thediseases set out in the fifth or sixth aspects of the invention above.

Typically, one or more of the compounds of structures (III) to (VIII)are used in the eighth aspect of the invention.

A ninth aspect of the present invention is a microbial culture or a foodor drink composition containing at least one microbial strain whichmicrobial strain is capable of producing one or more compounds of theformulae I or II from daidzein and/or glycitein.

Typically, said microbial strain produces one or both of compounds A andB.

Typically, the microbial strain is in the form of a purified culture,which may optionally be admixed and/or administered with one or moreother cultures which produce any one or more compounds of the formulae Ior II, more typically one or more of the compounds of structures (III)to (VIII).

A tenth aspect of the present invention provides a process for producinga compound of any one of formulae I or II by microbial fermentation ofdaidzein or glycitein with one or more microbial organisms selected fromthe group consisting of Lactobacilli; Clostridium perfingens; Bacteroidsincluding B. vulgatus, B. thetaiotaomicron, B. distasonis; Candidaalbicans and other yeast; Anaerobic cocci including Ruminococcus,Eubacterium, Peptostreptococcus (such as P. productus found in stools),Clostridium, Bifidobacteria (such as B. adolascentis, B. infantis, andB. longum), Peptococcus, Veillonella, Acidaminococcus, andStreptococcus; Anaerobic streptococci; Gram-negative facultativebacteria; Aeromonas such as A. hydrophila; Alcaligenes sp; Citrobactersp; Enterobacter sp including E. liquefaciens and E. aerogenes;Escherichia sp, E. coli; Hafnia sp; Klebsiella sp; Morganella sp such asM. morganii; Proteus sp; Pseudomonas sp; Providencia sp; Aerococcusviridans; Bacillus sp; Corynebacterium sp; Micrococcus sp such, as M.luteus; Nocardia sp; Pediococcus sp; Staphylococcus sp including S.aureus and S. epidermidis; Fusobacterium including F. gonidiaformans, F.mortiferum, F. necrogenes, F. necroforum and F. russii; Butyrivibriosuch as B. fibrisolvens; Actinomyces; Arachnia-Propionibacterium;Arthrobacter sp such as A. agilis, A. aurescens, A. pascens, A. oxydans,A. nicotinae and A. cummins; Brevibacterium sp such as B. epidermidis;and Microbacterium sp such as M. arborescens.

An eleventh aspect of the present invention provides a method for thetreatment, prophylaxis, amelioration, defence against, and/or preventionof menopausal syndrome including depression, anxiety, hot flushes, nightsweats, mood swings, and headache; osteoporosis; rheumatic diseases;atherosclerosis; premenstrual syndrome, including fluid retention,cyclical mastalgia, and dysmenorrhoea; coronary artery spasm; vasculardiseases including Reynauds Syndrome; Buergers Disease; migraineheadaches; hypertension; benign prostatic hypertrophy; all forms ofcancer including breast cancer, endometrial cancer, prostatic cancer,uterine cancer, ovarian cancer, testicular cancer, large bowel cancer;Alzheimers disease; inflammatory diseases including Crohns disease,inflammatory bowel disease, ulcerative colitis; baldness including malepattern baldness; psoriasis; acne; and diseases associated with oxidantstress including myocardial infarction, sunlight induced skin damage,arthritis, or cataracts, which method comprises administering to asubject a therapeutically effective amount of Factor 2 as previouslydefined, either alone or in association with one or morepharmaceutically acceptable carriers, diluents, adjuvants and/orexcipients.

According to a related twelfth aspect of the present invention there isprovided a method for the treatment, prophylaxis, amelioration, defenceagainst, and/or prevention of hormone-dependent conditions includinghormone dependent cancers such as breast cancer, hormone dependentcardiovascular disorder and hormone dependent menopausal disorderscomprising administering to a subject a therapeutically effective amountof Factor 2 as previously defined, either alone or in association withone or more pharmaceutically acceptable carriers, diluents, adjuvantsand/or excipients.

The invention also provides in a thirteenth aspect the use of Factor 2for the manufacture of a medicament for the treatment, prophylaxis,amelioration, defence against, and/or prevention of menopausal syndromeincluding depression, anxiety, hot flushes, night sweats, mood swings,and headache; osteoporosis; rheumatic diseases; atherosclerosis;premenstrual syndrome, including fluid retention, cyclical mastalgia,and dysmenorrhoea; coronary artery spasm; vascular diseases includingReynauds Syndrome; Buergers Disease; migraine headaches; hypertension;benign prostatic hypertrophy; all forms of cancer including breastcancer, endometrial cancer, prostatic cancer, uterine cancer, ovariancancer, testicular cancer, large bowel cancer; Alzheimers disease;inflammatory diseases including Crohns disease, inflammatory boweldisease, ulcerative colitis; baldness including male pattern baldness;psoriasis; acne; and diseases associated with oxidant stress includingmyocardial infarction, sunlight induced skin damage, arthritis, orcataracts.

A fourteenth aspect of the invention further provides the use of Factor2 for the manufacture of a medicament for the treatment, prophylaxis,amelioration, defence against, and/or prevention of hormone-dependentconditions including hormone dependent cancers such as breast cancer,hormone dependent cardiovascular disorder and hormone dependentmenopausal disorders.

A fifteenth aspect of the present invention provides a process for themanufacture of Compound A, said process including:

-   -   i) reacting 2-(p-methoxyphenyl)propionic acid with        1,3,4-trimethoxy benzene to obtain        2,4,5,4′-tetramethoxy-α-methyldesoxybenzoin; and    -   ii) demethylating said        2,4,5,4′-tetramethoxy-α-methyldesoxybenzoin to form        2,4,5,4′-tetrahydroxy-α-methyldesoxybenzoin.

A sixteenth aspect of the present invention provides a compound whenproduced by the process of the fifteenth aspect of the inventionoutlined above.

The present invention is based upon the identification of noveloestrogenic isoflavone metabolite compounds, exemplified by theisoflavonoid phytoestrogens of structures (III), (IV) and (V). Thesecompounds have been identified in the urine of the human adult consuminga diet rich in phytoestrogen content. While not wishing to be bound bytheory, it is postulated by the present inventor that the identificationof the compounds of structures (III), (IV) and (V) provides evidence forthe existence of a previously undiscovered pathway in the mode ofmetabolism of daidzein and/or glycitein.

The identification of the compounds of structures (III), (IV) and (V)observed for the first time in the urine of adult humans who ingestedsoya cake containing daidzein, genistein and glycitein provides evidenceto suggest that the compounds of structures (III), (IV) and (V) areproducts of microbial transformations of daidzein or glycitein. In viewof the fact that one of these metabolites, namely compound A, was foundin large amounts commensurate to the amount of daidzein ingestedcompared with glycitein appears that compounds A and B may also bemetabolites of daidzein after hydroxylation of ring A. The results ofKlus and Barz (1995) referred to above support this hypothesis sincethese authors demonstrated that a number of microbial species(Micrococcus, Arthrobacter, Brevibacterium) are capable of convertingdaidzein and glycitein to give Factor 2, the most probable precursor ofcompounds A and B.

The compounds of Formulae I and II of the present invention, all ofwhich include a vicinal diol substitution, show significant therapeuticactivity. In particular, it has been shown that compounds of theinvention inhibit the proliferation of MCF-7 and other ceils withoutsignificant enhancement of their growth at low concentrations. Thevicinal diol substitution is provided by at least one of the following:6,7-dihydroxy substitution in the benzopyran moiety of structure (I);3′,4′-dihydroxy substitution in the 3-phenyl substituent in structure(I); or 3,4-dihydroxy substitution and/or 3′,4′-dihydroxy substitutionin structure (II). It is speculated that it is the presence of thisvicinal diol substitution in the compounds of the invention whichconfers on them their surprisingly high biological activity.

DETAILED DESCRIPTION OF THE INVENTION

Compounds of the invention may be obtained by microbial fermentation ofsuitable naturally-occurring oestrogenic isoflavones, or by chemicalsynthesis.

For microbial fermentation, a plant source of naturally-occurringoestrogenic isoflavones is typically used.

Typically, plant sources for oestrogen isoflavone precursors of thecompounds of the invention are any leguminous plant including variousspecies of Acacia, ground nut, alfalfa, lentil and ground pea. Alsotypically, such plant sources include:

Trefolium species including parnassi, repens, pallescens, nigrescens,physodes, resupinatum, campestre, arvense, stellatum, cherleri,pignantii, alpestre, pratense, angustifolium, subterraneum andglomeratum, Medicago species including lupulina, falcata, orbicularis,polymorpha, disciformis, minima, and sativa, Cassia species includingoccidentalis and floribunda, Lupinus species including angustifolium andalbus, Vivia species including sativa and monantha and Galega speciesincluding officinalis, or mutant strains of any one the foregoing. Beanssuch as jumping bean, sword bean, broad bean, yam bean, kidney bean,soya bean and butter bean are also a favourable source of for oestrogenisoflavone precursors of the compounds of the invention. The oestrogenicisoflavones are mainly found in the leaves and fruit of the plant, andalso in the roots.

Typically, the compounds of interest which are secreted by microbialcultures or organisms are detected by GC-MS (gas chromatography-massspectrometry).

These organisms are used in microbial fermentation to produce compoundsof formulae I-II given above. Typically, the organisms are selected fromone of the following classes:

Lactobacilli; Clostridium perfingens; Bacteroids including B. vulgatus,B. thetaiotaomicron, B. distasonis; Candida albicans and other yeast;Anaerobic cocci including Ruminococcus, Eubacterium, Peptostreptococcus(such as P. productus found in stools), Clostridium, Bifidobacteria(such as B. adolescentis, B. infantis, and B. longum). Peptococcus,Veillonella, Acidaminococcus, and Streptococcus; Anaerobic streptococci;Gram-negative facultative bacteria: Aeromonas such as A. hydrophila;Alcaligenes sp; Citrobacter sp; Enterobacter sp including E.liquefaciens and E. aerogenes; Escherichia sp. E. coli; Hafnia sp;Klebsiella sp; Morganella sp such as M. morganii; Proteus sp;Pseudomonas sp; Providencia sp; Aerococcus viridans; Bacillus sp;Corynebacterium sp; Micrococcus sp such as M. luteus; Nocardia sp;Pediococcus sp; Staphylococcus sp including S. aureus and S.epidermidis; Fusobacterium including F. gonidiaformans, F. mortiferum,F. necrogenes, F. necroforum and F. russii; Butyrivibrio such as B.fibrisolvens; Actinomyces, Arachnia-Propionibacterium; Arthurobacter spsuch as A. agilis, A. aurescens, A. pascens, A. oxydans, A. nicotinaeand A. cummins; Brevibacterium sp such as B. epidermidis; andMicrobacterium sp such as M. arborescens.

Typically, non-pathogenic organisms selected from the above organismssuch as Micrococcus sp and Arthrobacter sp may be used directly in foodand/or drink compositions such as dairy formulations so as to providecompounds of the formulae of the invention. The drink/food compositionsalso need to contain a phytoestrogen source such as soya.

Microbial conversion of Daidzein and Glycitein to Factor-2 can beeffected using the following microbial organisms: Arthrobacter includingagilis, aurescens, pascens, oxydans, nicotinae, and cumminsii;Brevibacterium epidermidis (converts glycitein to Factor 2); Micrococcusluteus (converts glycitein to Factor 2), Microbacterium arborescens(converts daidzein lo Factor 2 & glycitein), Streptomyces sp roseolus(converts daidzein/glycitein to8,3′-dihydroxy-6,7,4-trimethoxyisoflavone or daidzein/glycitein to7,8,4′ & 7,3′,4′-trihydroxyisoflavones, depending on culture medium).The various microbial conversions are disclosed in detail in Klaus, K.and Barz, W.: Arch. Microbiol. 164 (1995) 428-434; Klaus. K.,Borger-Papendorf, G. and Barz, W.: Biochemistry 34(4) (1993) 979-981;Mackenbrock, K and Barz. W.: Naturforsch. 38c (1983) 708; Chimura, H. etal; J. Antibiot. 28 (1975) 619-626; Funayama, S. et al: J. Antibiot. 42(1989) 1350-1355 and Komiyama, K. et al: J. Antibiot. 42 (1989)1344-1349, the contents of all of which are incorporated herein byreference.

Without wishing to be bound by theory, the present inventor hypothesisesthat the metabolic pathways of catabolism of factor 2 obtained fromglycitein or daidzein are as shown in Scheme 1 below. Methylene unit(MU) values of the metabolites under the gas chromatographic conditionsdescribed in Example 1 are shown.

An alternative source of compounds of the present invention is chemicalsynthesis. Conveniently, Factor 2 or a naturally-occurring isoflavonesuch as glycitein may be utilised as starting material. Schemes 2A and2B demonstrate possible synthesis pathways of compounds of the inventionutilising glycitein as the starting material. In Scheme 2A, compounds 2and 4 may be obtained from glycitein by reduction with lithium aluminiumhydride as described in Example 1. A mixture of compounds 3, 5 and 7identified Scheme 2A may be obtained from compound 8 as shown in Scheme2B.

Compounds of the equol or dehydroequol series may also be prepared fromthe corresponding dihydroisoflavone (exemplified by compound 2 in Scheme2a) by reduction of the carbonyl and dehydration of the resultingalcohol to give a compound of the dehydroequol series, and optionallycatalytically hydrogenating the double bond in the pyran ring to yieldthe corresponding compound of the equol series.

Unlike the isoflavonoid metabolites of the daidzein and genisteinseries, those of glycitein have the synthetic advantage that the vicinalhydroxyl groups in the A-ring allow a number of protective functionalgroups such as the ketals and boronates to be formed easily. In thescheme (Scheme 3) below, the synthesis of compounds of Formula II isdemonstrated using a 1,2,4-benzenetriol substrate which, has beenprotected as an n-butyl boronate derivative formed using commerciallyavailable n-butylboronic acid according to methods adopted in similarprotective reactions [Joannou, G. E. and Reeder, A. Y., Steroids 6111-17. (1996)]. Other alkyl boronates can be used.

Synthesis of 4′ Methoxy-5-Hydroxy-O-Dma and Similar Molecules

In Scheme 3, one of R₁ and R₂ is H, OH or OCH₃ and the other is OH orOCH₃. For the synthesis of compounds of Formula II in which is OCH₃, thefree hydroxyl group in the boronate intermediate shown above may bemethylated, for example by reaction with methyl iodide or methylsulfate. It will be appreciated that when R₁ or R₂ is OH, it may requireprotection. When R₁ and R₂ are both OH, they may be protected as acyclic boronate, ketal or carbonate.

Instead of using n-butylboronic acid, formation of protective functionalgroups may alternatively be achieved using cyclic carbonates, cyclicacetals or ketals as shown in Scheme 4A below. Partial methylation canalso be used as shown in Scheme 4B below. Other protective groups forcatechols are described in Chapter 3 of Greene, T. W. and Wuts, P. G.M.: Protective Groups in Organic Synthesis (2^(nd) Edition) (1991) JohnWiley & Sons, Inc. USA; the disclosure of which is incorporated hereinby reference. Compounds of the invention in which R₁ is H and R₂ is OHor OCH₃ or in which R₁ and R₂ are both OH or OCH₃ may be synthesised byanalogous procedures to that shown in Scheme 4A but starting with2-(3-methoxyphenyl)propanoic acid or 2-(3,4-dimethoxyphenyl)propanoicacid instead of the corresponding 4-methoxyphenyl derivative. Similarly,compounds of formula (II) in accordance with this invention, in whichone of R₃ and R₄ is H, may be prepared by an analogous procedurebeginning with reaction of resorcinol or hydroquinone, suitablyprotected, with poly phosphoric acid.

Furthermore formation of cyclic protective groups such as thosedescribed above will allow the synthesis of a number of the isoflavonoidcompounds proposed which are normally difficult to obtain syntheticallyas in the case of the tetrahydro, dehydro and equol analogues ofglycitein or its demethylated analogues. A schematic representation(Scheme 5) is given below using 4′,6,7-trihydroxyglycitein as anexample.

When necessary, hydroxyl groups in the compounds shown in Schemes 1-5above, may be methylated and/or protected and deprotected, to give othercompounds of formula I or II. Suitable protecting groups are describedin the work of Greene and Wills referenced above.

Compound A may be prepared by the following synthetic scheme 6:

Compound B and related compounds of formula (I) or (II) may be preparedas shown in Scheme 7, in which R is CH₃, R′ is C₂H₅, R₁-R₄ are each H,OH or OCH₃, and R′₁-R′₄ are each H or OH, subject to the proviso that inthe final product of formula (I) or (II) R′₁ and R′₂ are both OH and/orR′₃ and R′₄ are both OH.

In the above Scheme 7, the base is typically an organic amine, such asdimethylamine, or an alkali metal hydroxide, carbonate or bicarbonate.

In the synthesis of 4′,6,7-trihydroxyisoflavone(5-deoxydihydroglycitein) shown in Scheme 7 above, the two intermediatesobtained in the penultimate step prior to the demethylation with BBr₃arc not easily separated. However, it was found that a simplerecrystallization procedure using methanol/water provided a quick methodof separation and purification of the two intermediates. A similarprocedure may be applied to the isolation of the methylated precursorsof daidzein and genistein, namely formononetin and biochanin A which arepresent in clover and soya. Complete methylation of formononetin andbiochanin A may further enhance the process of recrystallization ofthese two isoflavonoid precursors. Isolated formononetin or its fullymethylated analogue can be used as a substrate for the chemical ormicrobial transformations to give Factor 2 or any of the compounds ofFormula I or II defined above.

As an example, formononetin or its methylated analogue may be isolatedfrom a rich source such as clover or soya for subsequent microbialtransformation to Factor 2 or a compound of formula I or II.Alternatively, isolates of clover extracts containing is formononetinand daidzein may be fermented to produce Factor 2 or its methylatedanalogue for extraction with water and/or an organic solvent. As afurther possibility, Factor 2 and compounds of formula I or II, may beobtained by chemical transformation of formononetin, daidzein, glyciteinor other naturally-occurring isoflavones as described in more detailabove.

The compounds of the formulae I or II, or Factor 2, may be administeredin a manner as is generally known in the art. The dosage utilised willdepend upon a number of factors including the specific application, thecondition being treated, the mode of administration, the state of thesubject, the route of administration and the nature of the particularcompound used.

Typically, a daily dose amount of a compound of the invention, such asany of the compounds of structures (III) to (VIII) which is required ina therapeutic treatment according to the invention, is in the range of0.1 mg to 2 g; more typically from 0.5 mg to 1 g: even more typicallyfrom 50 mg to 500 mg; most typically from 50 to 250 mg.

In the production of a pharmaceutical composition of the presentinvention any one or more of the compounds of formulae I or II, orFactor 2, is/are typically admixed with one or more pharmaceuticallyacceptable carriers, adjuvants, diluents and/or excipients as are wellknown in the art.

The carrier must, of course, be acceptable in the sense of beingcompatible with any other ingredients in the composition and must not bedeleterious to the subject. The carrier or excipient may be a solid or aliquid, or both, and is preferably formulated with the compound as aunit-dose, for example, a tablet, which may contain from 0.5% to up to100% by weight of the active compound.

Typically, one or more of the compounds of structures (III) to (VIII)may be incorporated in the compositions of the invention, which may beprepared by any of the well known techniques of pharmacy consistingessentially of admixing the components, optionally including one or moreaccessory ingredients.

The compositions of the invention are typically formulated to includethose suitable for rectal, optical, oral, buccal, parenteral (forexample, subcutaneous, intramuscular, intradermal, or intravenous) andtransdermal administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated and on the nature of the particular active compound which isbeing used.

For parenteral administration, the compound(s) of the invention may beprepared in sterile aqueous or oleaginous solution or suspension.Suitable non-toxic parenterally acceptable diluents or solvents includewater, Ringer's solution, isotonic salt solution, 1,3-butanediol,ethanol, propylene glycol or polyethylene glycols in mixtures withwater. Aqueous solutions or suspensions may further comprise one or morebuffering agents. Suitable buffering agents include sodium acetate,sodium citrate, sodium borate or sodium tartrate, for example.

Compositions of the invention may be prepared by means known in the artfor the preparation of compositions (such as in the art of preparingveterinary and pharmaceutical compositions) including blending,grinding, homogenising, suspending, dissolving, emulsifying, dispersingand where appropriate, combining or mixing of the compound(s) of any ofFormulae I or II, or Factor 2 together with the selected excipient(s),carrier(s), adjuvant(s) and/or diluent(s).

Compositions formulated as suitable for oral administration may bepresented in discrete units, such as capsules, cachets, lozenges, ortablets, each containing a predetermined amount of the preferred activecompound; as a solution or a suspension in an aqueous or non-aqueousliquid; as a powder or granules; or as an oil-in-water or water-in-oilemulsion. For example, compressed tablets may be prepared by compressingany one or more compounds of formulae I or II, or Factor 2, in afree-flowing form, such as a powder or granules, optionally mixed with abinder, lubricant inert diluent, and/or surface active/dispersingagent(s). Moulded tablets may be made by moulding, in a suitablemachine, a powdered compound of any one of formulae I or II, or Factor2, moistened with an inert liquid binder.

Solid forms for oral administration may contain pharmaceutically orveterinarily acceptable binders, sweeteners, disintegrating agents,diluents, flavourings, coating agents, preservatives, lubricants and/ortime delay agents. Suitable binders include gum acacia, gelatin, cornstarch, gum tragacanth, sodium alginate, carboxymethylcellulose orpolyethylene glycol. Suitable sweeteners include sucrose, lactose,glucose, aspartame or saccharine. Suitable disintegrating agents includecorn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum,bentonite, alginic acid or agar. Suitable diluents include lactose,sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate,calcium silicate or dicalcium phosphate. Suitable flavouring agentsinclude peppermint oil, oil of wintergreen, cherry, orange or raspberryflavouring. Suitable coating agents, include polymers or copolymers ofacrylic acid and/or methacrylic acid and/or their esters, waxes, fattyalcohols, zein, shellac or gluten. Suitable preservatives include sodiumbenzoate, vitamin E. alpha-tocopherol, ascorbic acid, methyl paraben,propyl paraben or sodium bisulfite. Suitable lubricants includemagnesium stearate, stearic acid, sodium oleate, sodium chloride ortalc. Suitable time delay agents include glyceryl monostearate orglyceryl distearate.

Liquid forms for oral administration may contain, in addition to theabove agents, a liquid carrier. Suitable liquid carriers include water,oils such as olive oil, peanut oil, sesame oil, sunflower oil, saffloweroil, arachis oil, coconut oil, liquid paraffin, ethylene glycol,propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol,glycerol, fatty alcohols, triglycerides or mixtures thereof.

Suspensions for oral administration may further comprise dispersingagents and/or suspending agents. Suitable suspending agents includesodium carboxy-methylcellulose, methylcellulose,hydroxypropylmethyl-cellulose, polyvinyl-pyrrolidone, sodium alginate orcetyl alcohol. Suitable dispersing agents include lecithin,polyoxyethylene esters of fatty acids such as stearic acid,polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate,polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate andthe like.

The emulsions for oral administration may further comprise one or moreemulsifying agents. Suitable emulsifying agents include dispersingagents as exemplified above or natural gums such as gum acacia or gumtragacanth.

For parenteral administration, the active compound(s) of Formulae I orII or Factor 2 may be prepared in sterile aqueous or oleaginous solutionor suspension. Suitable non-toxic parenterally acceptable diluents orsolvents include water. Ringer's solution, isotonic salt solution, 5%dextrose in water, buffered sodium or ammonium acetate solution,1,3-butanediol, ethanol, propylene glycol or polyethylene glycols inmixtures with water. Aqueous solutions or suspensions may furthercomprise one or more buffering agents. Suitable buffering agents includesodium acetate, sodium citrate, sodium borate or sodium tartrate, forexample. These preparations suitable for parenteral administration, arepreferably administered intravenously, although administration may alsobe effected by means of subcutaneous, intramuscular, or intradermalinjection. Aqueous solutions for parenteral administration are alsosuitable for administration orally or by inhalation.

Typical parenterally administered preparations may conveniently beprepared by admixing one or more of the compounds of structures (III) to(VIII) with water or a glycine buffer and rendering the resultingsolution sterile and isotonic with the blood. Injectable formulationsaccording to the invention generally contain from 0.1% to 70% w/v ofactive compound and are typically administered at a rate of 0.1ml/minute/kg.

For rectal administration, the compound(s) of Formulae I or II or Factor2 is suitably administered in the form of an enema or unit dosesuppository. A suitable suppository may be prepared by mixing the activesubstance with a non-irritating excipient which is solid at ordinarytemperatures but which will melt in the rectum. Suitable such materialsare cocoa butter, waxes, fats, glycerol, gelatin and polyethyleneglycols. Suitable enemas may comprise agents as exemplified above withreference to forms for topical administration.

Suitably, an inhalation spray comprising a compound(s) of Formulae I orII or Factor 2 will be in the form of a solution, suspension or emulsionas exemplified above. The inhalation spray composition may furthercomprise an inhalable propellant of low toxicity. Suitable propellantsinclude carbon dioxide or nitrous oxide.

The pharmaceutical composition may contain pharmaceutically acceptablebinders, diluents, disintegrating agents, preservatives, lubricants,dispersing agents, suspending agents and/or emulsifying agents asexemplified above. The veterinary composition may contain veterinarilyacceptable binders, diluents, disintegrating agents, preservatives,lubricants, dispersing agents, suspending agents and/or emulsifyingagents as exemplified above.

The invention includes compositions which are used for topicalapplication which may be a cream, ointment, paste, solution, emulsion,lotion, milk, jelly, gel, spray, aerosol, oil, stick, roll-on orsmooth-on, wherein the active compound comprises up to about 90%, moretypically 10%, by weight of the composition, even more typically fromabout 0.1% to about 5% by weight, for example 3.5% by weight, even moretypically from 0.5% to 2% w/w, and the compositions include topicallysuitable carriers, diluents, excipients, adjuvants and other additives.

Illustrative of pharmaceutically or cosmetically topically acceptablecarriers or diluents are demineralized or distilled water; salinesolution; vegetable based oils such as peanut oil, safflower oil, oliveoil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil;silicone oils, including polysiloxanes, such as methyl polysiloxane,phenyl polysiloxane and methylphenyl polysiloxane; volatile silicones;mineral oils such as liquid paraffin, soft paraffin or squalane;cellulose derivatives such as methyl cellulose, ethyl cellulose,carboxymethylcellulose, sodium carboxymethylcellulose orhydroxypropyl-methylcellulose; lower alkanols, for example ethanol oriso-propanol; lower aralkanols; lower polyalkylene glycols or loweralkylene glycols, for example polyethylene glycol, polypropylene glycol,ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin;fatty acid esters such as isopropyl palmitate, isopropyl myristate orethyl oleate; polyvinylpyrrolidone; agar; carrageenan; gum tragacanth orgum acacia, and petroleum jelly. Typically, the carrier or carriers willform from 10% to 99.9% by weight of the composition.

Adjuvants typically include emollients, emulsifiers, thickening agents,preservatives, bacteriocides and buffering agents.

Emollients suitable for inclusion In a topical composition of theinvention include fatty esters such as isopropyl myristate, cetylacetate, diisopropyl adipate or C₁₂-C₁₅ alcohol benzoates: fattyalcohols such as lauryl alcohol, myristyl alcohol, cetyl alcohol,stearyl alcohol or cerostearyl alcohol; mineral and vegetable oils suchas, aloe vera and jojoba oil; lecithin: Vitamin E; lanolin; sorbitol andglycerin. Typically, the emollient or emollients will form from 10% to99.9% by weight of the composition.

Suitable thickening agents include sodium stearate, calcium stearate,magnesium stearate, calcium palmitate and magnesium palmitate, dextran,dextrins, starch and starch products, gelatin, cellulose derivatives asexemplified above, collagen, water soluble polymers such as carboxyvinylpolymer, polyvinyl alcohol or polyvinyl acetate, pectin, xanthan gums,bentonite, hyaluronic acid, fumed silica and the like. Typically, thethickening agent or agents will form from 0.1% to 20% by weight of thecomposition.

Typical preservatives include ascorbic acid and its salts, erythorbicacid and its salts, ethyl and iso-propyl p-hydroxybenzoates,benzalkonium chloride, benzyl alcohol, phenyl ethanol and glydantchlorobutanol. Typically, the preservative or preservatives will formfrom 0.1% to 12% by weight of the composition.

Suitable buffering agents are salts of boric, acetic, phosphoric,citric, malic, silicic acids and the like, for example sodium citrate,sodium bicarbonate, sodium acetate and sodium phosphate. Additionally oralternatively, the free acids may be used, together with an alkali suchas sodium hydroxide, sodium carbonate, sodium bicarbonate, potassiumhydroxide, potassium carbonate or potassium bicarbonate. Typically, thebuffering agent or agents will form from 0.1% to 20% by weight of thecomposition.

Emulsifiers may also be included in a topical composition of theinvention. Illustrative nonionic emulsifiers include fatty acids such asoleic acid, stearic acid and palmitic acid: esters of lactic acid,tartaric acid, ascorbic acid or citric acid; polyalkylene glycol esterssuch as polyoxyethylene glycol monostearates, polyoxyethylene glycolmonolaurates; polyoxyethylene glycol distearates or polyoxyethyleneglycol dilaurates; polyalkylene glycol ether derivatives of aliphatic orcycloaliphatic alcohols such as polyoxyethylene nonylphenol ether,polyoxyethylene cetyl ether or polyoxyethylene stearyl ether: hexitanesters, for example sorbitan monolaurate, sorbitan monooleate, sorbitandistearate, sorbitan tristearate, sorbitan dilaurate or sorbitantrilaurate; fatty esters such as glyceryl monostearate, ethylene glycolmonostearate, propylene glycol monostearate or butylene glycolmonostearate; sorbitol and ethoxylated sorbitol esters of fatty acidssuch as polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitanmonolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylenesorbitan distearate, polyoxyethylene sorbitan dilaurate, polyoxyethylenesorbitan dioleate, polyoxyethylene sorbitan tristearate, polyoxyethylenesorbitan trilaurate or polyoxyethylene sorbitan trioleate; long-chainalcohols such as lauryl, myristyl, stearyl, oleyl, cetyl or cerostearylalcohol; polysaccharides such as starch and starch derivative, cellulosederivatives as exemplified above, agar, tragacanth, acacia and alginicacid; and steroidal derivatives such as lanolin alcohols or ethoxylatedlanolin alcohols, and beeswax. Illustrative ionic surfactants includetriethanolamine and amine soaps such as triethanolamine stearate;anionic soaps such as calcium or magnesium salts of stearic acid orpalmitic acid; fatty alcohol sulfates, for example sodium laurylsulfate; alkyl or aralkyl sulfanates such as sodium sulfosuccinates orsodium dodecylbenzenesulfonate; quaternary ammonium salts containing atleast one long-chain alkyl group as N-substituent, for example stearyltrimethylammonium chloride, and phosphate esters of polyalkyleneglycols. Typically, the emulsifier or emulsifiers will form from 0.1% to99% by weight of the composition.

The topical compositions of the invention may further include asunscreen. Suitable sunscreens include opacifiers such as titaniumdioxide or zinc oxide; p-aminobenzoic acid, isobutyl p-aminobenzoate,glyceryl p-aminobenzoate, or N-substituted derivatives of p-aminobenzoicacid such as isoamyl p-dimethylaminobenzoate, pentylp-dimethylaminobenzoate, octyl p-dimethylaminobenzoate or ethyl4-[bis(2-hydroxypropyl)amino]benzoate; 2-hydroxy-1,4-naphthoquinone;octocrylene; octyl p-methoxycinnamate or 2-ethoxyethylp-methoxycinnamate: salicylate esters such as octyl salicylate,homomenthyl salicylate or 2-[bis(2-hydroxyethyl)-amino]ethyl salicylate;oxybenzone and methyl anthranilate. Typically, the sunscreen orsunscreens will form from 0.1% to 10% by weight of the composition.

Additionally, it will be understood that the topical compositions of theinvention may include suitable colouring agents and/or perfumes wellknown in the art. Typical examples of suitable perfuming agents areprovided in S. Arctander, “Perfume and Flavor Chemicals”, Montclair,N.J., 1969.

Formulations suitable for transdermal administration are typicallypresented as discrete patches adapted to remain in intimate contact withthe epidermis of the recipient for a prolonged period of time. Suchpatches suitably contain at least one compound of formulae I or II, orFactor 2, preferably one or both of compounds A and B. as an optionallybuffered aqueous solution of, for example, 0.1 M to 0.5 M concentrationwith respect to the said active compound. More typically, one or both ofcompounds A and B are present in a concentration of 0.1-0.3 Mconcentration.

The active compounds of formulae I or II may be provided in the form offood and/or drink compositions, such as being added to, admixed into,coated or combined with a food or drink product.

Typically, food and drink compositions of the present invention aredairy based. More typically, one or more of compounds of structures(III) to (VIII) are combined or otherwise formulated into a dairy basedfood or drink product such as a milk drink or supplement, and a chilledor frozen dairy product such as a dairy based dessert.

Therapeutic methods, uses and compositions may be for administration tohumans or animals, including domestic animals, birds (includingchickens, turkeys, ducks), livestock animals (such as cattle, sheep,pigs and goats) and the like.

It will be appreciated that the examples referred to above areillustrative only and other suitable carriers, diluents, excipients andadjuvants known to the art may be employed without departing from thespirit of the invention.

Embodiments of the invention will now be described with reference to thefollowing non-limiting Examples.

EXAMPLE 1

5-hydroxy-O-demethylangolensin (Compound A)[1-(2,4,5-trihydroxyphenyl)-2-(4′-hydroxyphenyl)-propan-1-one]

1. As Product of Lithium Aluminium Hydride Reduction Reaction fromGlycitein

Glycitein (20.16 mg, 0.75×10⁻⁷ mol) was weighed out and dried undervacuum. The dried glycitein was dissolved in anhydrous THF (˜3.0 ml) andto this solution 10 eq of LiAlH₄ (1.0 M in ether) was added dropwise atroom temperature. The reaction was allowed to stir at room temperatureovernight, then refluxed for 5 hr. After workup the solution wasfiltered through celite using methanol. The filtrate was concentratedand analysed by GC and HPLC (MeOH/H₂O 40:60). Among the productsseparated by GC those at MU 25.69 and 28.65 were the major ones. Afterisolation of the two major products by preparative HPLC, these wereanalysed by GC-MS characterising them as derivatives of Compounds A andB respectively. Demethylation of these products was achieved by borontribromide in dichloromethane at room temperature for three daysaccording to Bannwart C et al., (Finn. Chem. Lett. 1984, Vol 11, p 120).In performing the GC-MS, a 30 metre SE30 capillary column was used withtemperature program of 200-230° C. at increments of 2° C./min, and230-280° C. at increments of 10° C./min. The carrier gas was helium.

2. As a Product of Acylation Reaction

Step 1: Formation of 2,4,5,4′-tetramethoxy-α-methyldesoxybenzoin. To amixture of 2-(p-methoxyphenyl)propionic acid (0.20 g, 1.11 mmol) andpolyphosphoric acid (5 gm), 1,3,4-trimethoxy benzene (0.186 g, 1.11mmol, 0.166 ml) was added. The mixture was allowed to heat to 75° C.while stirring for 6 hours. TLC (30% EtOAc:Hexane) and gaschromatography (GC) and gas chromatography-mass spectrometry (GC-MS)analyses confirmed the presence of two major products with MU values of24.68 and 25.01 (ratio 1:4), Chromatography on silica column (30%EtOAc:Hexane) allowed the isolation of the two products. Product MU24.92 was isolated as a crystalline low melting solid. NMR data andGC-MS data confirmed the above structure. A 42% and 11% yield wasobtained for products MU 25.01 and MU 24.68 respectively.

Step 2: Formation of 2,4,5,4′-tetrahydroxy-α-methyldesoxybenzoin. Theproduct 2,4,5,4′-tetramethoxy-α-methyldesoxybenzoin (MU 25.01; 0.063 g)obtained from Step 1 above was dissolved in anhydrous dichloromethane(30.0 mL) and boron tribromide (0.271 g, 1.08 mmol) was added to thesolution. The mixture was allowed to stir at room temperature for 24hours under nitrogen. TLC (30% EtOAc:hexane) established the presence ofa single product which on GC analysis as the trimethylsilyl ether gave asingle peak at MU 26.01. After workup with ice/water the product wasextracted with diethyl ether, washed with water, dried and concentratedto give a crude yellow oil, which by NMR and GC-MS data was confirmed tobe 2,4,5,4′-tetrahydroxy-α-methyldesoxybenzoin.

Mass Spectra Data (EIMS: electron ionisation; CIMS; Chemical ionisation;High resolution; HR)

HR: 274.084267, theoretical 274.084267.

EIMS: m/z (% rel int) 274 [M]+ (14), 153(100); 121(29), 77(8).

EIMS as the tetra-trimethylsilyl derivative: 562(1.6): 547(4.7);457(1.6): 369(100); 281(6.7); 193(5.4); 147(2.7).

CIMS as the tetra-trimethylsilyl derivative: M+b 1=563(75); 547(59):491(15); 370(31); 369(100); 193(22).

NMR Data

¹H n.m.r.

(Acetone-d6, 2.05 ppm) δ 1.39 (3H, d, J=7.2 Hz, CH₃), 4.62 (1H, q, J=7.2Hz, CH), 6.29 (1H, s, ArH-3), 6.75 (2H, d, J=9.2 Hz, ArH-3′,5′), 7.17(2H, d, J=9.2 Hz, ArH-2′,6′), 7.33 (1H, s, Ar-6) 8.73

¹³C n.m.r.

(Acetone-d6, ppm) 1873, 45.59, 103.05, 110.845, 115.38, 115.58, 128.51,132.96, 137.60, 153.86, 156.25, 159.85, 204.77.

UV: λ_(max)=283 nm

EXAMPLE 2

5-deoxydihydroglycitein (Compound B)

Compound B was obtained in a series of reactions as illustrated inScheme 7, involving an acylation reaction, formation of an α-alkenylketone and cyclisation/demethylation. In brief,2,4,5-trimethoxyphenyl-4′-methoxybenzyl ketone was obtained as anintermediate in an acylation reaction using 1,2,4-trimethoxybenzene (5.9mmol), 4-methoxyphenylacetic acid (5.9 mmol) and polyphosphoric acid (17gm) after beating at 70° C. for one hour with mechanical stirring.Potassium carbonate was then added to the reaction for another one andhalf hours. The crude product was purified by recrystallization fromethyl acetate and light petroleum to give light yellow crystals (75%yield). The α-alkenyl ketone was subsequently obtained by a modificationof Gandhidasan's method (Gandhidasan R et al., Synthesis, 1982, 1110).In brief, to a suspension of 2,4,5-trimethoxyphenyl-4′-methoxybenzylketone in ethanol, paraformaldehyde and N,N-dimethylamine was added andthe mixture was allowed to reflux while heated for one hour. When thereaction was complete, the precipitate was filtered and the filtrate wasconcentrated in vacuo, after which the residue was dissolved in ethylacetate and washed with water. The organic layer was dried withmagnesium sulphate and filtered, and the solvent was removed to give thecrude product. On purification by flash chromatography two compoundswere obtained in 57% yield. Fractional recrystallization of the mixturegave 1-(4-methoxyphenyl)-1-(2,4,5-trimethoxybenzoyl)ethylene as themajor product (˜41%) andα-ethoxymethyl-2,4,5-trimethoxyphenyl-4′-methoxybenzyl ketone as theminor product (˜17%). The method provided the best yields when 1%potassium bicarbonate is used instead of the dimethylamine in themethylation step.

When sodium hydroxide was used instead the percentage yield was lowernamely 38% and 14% respectively for these two products. The desireddihydro product Compound B (6,7,4′-trihydroxyisoflavone) was finallyobtained by demethylation of1-(4-methoxyphenyl)-1-(2,4,5-trimethoxybenzoyl)ethylene using borontribromide in dichloromethane at room temperature for three daysaccording to Bannwart C et al., Finn. Chem. Lett. 11 120 (1984) followedby cyclisation of the resulting brominated intermediate by sodiumacetate in methanol.

In the formation of the α-alkenyl ketone in the absence of a base thereaction will not proceed and the starting material will remainunchanged. The good yield of this method provides a good chemical methodfor the synthesis of a number of the dihydro derivatives of daidzein,genistein or glycitein.

Mass Spectra Data (EIMS electron ionisation; CIMS Chemical ionization;High resolution HR)

Compound B: HR: 272.0673 theoretical 272.0673

EIMS: m/z (% rel. int) 272 [M]+ (31), 244(9); 168(7); 153(100); 120(40);107(27); 91(11).

CIMS: 301 M+29(14); 273 M+1(52); 257(37); 137(23); 97(17); 83(45);71(100).

EIMS as the tri-trimethylsilyl derivative: MU 28.48, MW 488; 488(14);473(7); 369(30); 296(100); 281(9); 192(27); 177(24); 147(9).

NMR Data

¹H n.m.r. (Acetone-d6) δ 2.05 ppm (1H, dd, J_(3.2eq)=5.0 Hz,J_(3.2ax)=9.5 Hz, H-3), 4.14 (1H, dd, J_(2ax,2eq)=9.7 Hz, JJ_(2ax,3)=9.6 Hz, H_(2ax)), 4.99 (1H, dd, J_(2eq,2ax)=9.8 Hz, J2eq,3=4.9 Hz, H_(2eq)), 6.38 (1H, s, ArH-8), 6.82 (2H, d, J=8.6 Hz,ArH-3′,5′), 7.27 (2H, d, J=8.6 Hz, ArH-2′,6′), 7.46 (1H, s, ArH-5).

¹³C n.m.r. (Acetone-d6, 29.8 ppm) δ 33.5, 54.6, 103.9, 111.9, 116.6,128.4, 129.3, 138.78, 155.2, 158.0, 160.4, 201.8.

UV: λ_(max)=284 nm

EXAMPLE 3

1-(2,4-dihydroxyphenyl)-2-(3′,4′-dihydroxyphenyl)-1-oxo-2-propene(3′-hydroxy-O-demethyldehydroangolesin); Structure (VI)1. 1-(2,4-dihydroxyphenyl)-2-(3′,4′-dihydroxyphenyl)-1-oxo-ethane

A mixture of 1,3-dimethoxybenzene (2.00 g, 14.47 mmol) and3,4-diemethoxyphenylacetic acid (2.84 g, 14.47 mmol) in polyphosphoricacid was heated at 80° C. for 2 hours. After cooling, the mixture waspoured onto ice water and the water was extracted with ethyl acetate (50mL). The combined organic phases were washed with water, sodiumbicarbonate solution and water and dried over anhydrous magnesiumsulfate. Evaporation of the solvent gave light yellow crystals of1-(2,4-dihydroxyphenyl)-2-(3′,4′-dihydroxyphenyl)-1-oxo-ethane whichwere purified by recrystallisation.

2. 1-(2,4-dimethoxyphenyl)-2-(3′,4′-dimethoxyphenyl)-1-oxo-2-propene and1-(2,4-dimethoxyphenyl)-2-(3′,4′-dimethoxyphenyl)-1-oxo-3-ethoxy-propane

A mixture of the product of step 1 (3.504 g, 11.08 mmol), 95%paraformaldehyde (1.275 g, 46.66 mmol) and N,N-dimethylamine (5.6 mL,46.66 mmol) in ethanol (58 mL) was heated under reflux for one hour.Then potassium carbonate (1.612 g, 11.67 mmol) was added to the mixtureand heating under reflux was continued for a further three hours afterwhich the precipitate was removed by filtration and the solvent wasremoved under reduced pressure. The residue was dissolve in ethylacetate and the solution was washed with water. 0.2 M HCl and water,dried over magnesium sulfate and concentrated to give a yellow oil.1-(2,4-dimethoxyphenyl)-2-(3′,4′-dimethoxyphenyl)-1-oxo-2-propene wasseparated from1-(2,4-dimethoxyphenyl)-2-(3′,4′-dimethoxyphenyl)-1-oxo-3-ethoxy-propaneby column chromatography with a mobile phase of 40% ethyl acetate inhexane.

3. 1-(2,4-dihydroxyphenyl)-2-(3′,4′-dihydroxyphenyl)-1-oxo-2-propene

0.406 g (1.08 mmol) of1-(2,4-dimethoxyphenyl)-2-(3′,4′-dimethoxyphenyl)-1-oxo-3-ethoxy-propanewere reacted with boron tribromide (10.84 mmol) in 22 mL dichloromethanefor three days by the method of Bannwart C. et al., Finn. Chem. Lett. 11120 (1984). Workup and chromatography of the reaction product afforded1-(2,4-dihydroxyphenyl)-2-(3′,4′-dihydroxyphenyl)-1-oxo-2-propene as theminor product and1-(2,4-dihydroxyphenyl)-2-(3′,4′-dihydroxyphenyl)-1-oxo-3-bromo-propaneas the major product.

Mass spectral data (electron impact) for1-(2,4-dihydroxyphenyl)-2-(3′,4′-dihydroxyphenyl)-1-oxo-2-propene astetra-TMS derivative: m/z (% relative intensity) at 209(10), 267(4.5),281(100), 545(20), 560(23).

EXAMPLE 4

7-hydroxy-(3′,4′-dihydroxyphenyl)-2,3-dihydroisoflavone(3′-hydroxy-dihydro-daidzein); Structure (VII)

0.157 g of1-(2,4-dihydroxyphenyl)-2-(3′,4′-dihydroxyphenyl)-1-oxo-3-bromo-propane,the major product of step 3 in Example 3, and about 2 molar equivalentsof sodium acetate ware mixed with 88 mL of methanol and heated at about60° C. for 4 hours. After cooling, the mixture was acidified to pH 5 andthe methanol was removed under reduced pressure. The residue wasdissolved in ethyl acetate (50 mL) and the solution was washed withwater and concentrated. The crude product was separated by columnchromatography to yield approximately equal amounts of1-(2,4-dihydroxyphenyl)-2-(3′,4′-dihydroxyphenyl)-1-oxo-2-propene and7-hydroxy-(3′,4′-dihydroxyphenyl)-2,3-dihydroisoflavone.

Mass spectral data (electron impact) for7-hydroxy-(3′,4′-dihydroxyphenyl)-2,3-dihydroisoflavone as tri-TMSderivative: m/z (% relative intensity) at 192(7.2), 281(100), 473(6.6),488(17).

EXAMPLE 5

1-(2,4-dihydroxyphenyl)-2-(3′,4′-dihydroxyphenyl)-1-oxopropane;Structure (V)

The title compound was obtained by catalytic hydrogenation of1-(2,4-dihydroxyphenyl)-2-(3′,4′-dihydroxyphenyl)-1-oxo-2-propeneobtained as in Example 3 or Example 4. To a solution of1-(2,4-dihydroxyphenyl)-2-(3′,4′-dihydroxyphenyl)-1-oxo-2-propene inmethanol was added palladium on carbon, and hydrogen gas was bubbledvigorously through the solution for ten minutes. Removal of the catalystand evaporation of the solvent afforded the title compound.

Mass spectral data (electron impact) for1-(2,4-dihydroxyphenyl)-2-(3′,4′-dihydroxyphenyl)-1-oxopropane astetra-TMS derivative: m/z (% relative intensity) at 209(5.8), 281(100),369(2.4), 457(1.2), 459(1.3), 547(4.0), 562(1.2).

EXAMPLE 6

1-(2,4,5-trihydroxyphenyl)-2-(4-hydroxyphenyl)-1-oxo-2-propene(5-hydroxy-2-dehydro-O-Dma); Structure (VIII)

This compound was prepared as shown in Scheme 7 utilising methodologyanalogous to dial described in Example 3.

Mass spectral data (electron impact) for1-(2,4,5-trihydroxyphenyl)-2-(4-hydroxyphenyl)-1-oxo-2-propene astetra-TMS derivative: m/z (% relative intensity) at 147(40), 281(28),369(63), 370(20), 545(94), 546(46), 560(100), 561(50), 562(27).

EXAMPLE 7 Bacterial sp and Culture Conditions:

The standard incubation assays of bacteria (100 mg wet wt) withisoflavone substrates (5×10⁻⁵ M), the composition of the mineral saltmedium and the isolation of the transformation products from the mediumwere essentially as described according to Klus,

25 K. et al., Arch. Microbiol. 164 428-434 (1995). The mineral mediumand micronutrients were used according to Pfennig and Lippert (1966). Insummary Bacterial sp were cultivated on Merck Standard I nutrient agarand for incubation experiments for 15 hr in 100 ml Merck Standard Inutrient broth. Prior to incubation the bacteria were washed twice with200 ml Kpi buffer (0.05 M, pH 7.5). After centrifugation (10,000 g, 15min) 100 mg bacteria (fr. Wt) were inoculated in 5 ml mineral medium and50 μl substrate solution (DMSO-MeOH, 1:10) was applied to the bacterialculture. Substrate concentration was 5×10⁻⁵. The cultures were incubatedin culture tubes (200×16 mm) in an orbital shaker at 200 rpm. 30° C.

EXAMPLE 8 Effects of Isoflavonoid Phytoestrogens on the Induced Growthof MCF-7 Cells and Other Cells.

Compound A was compared with genistein to test the cell viability ofMCF-7 cells. Genistein was known, prior to this invention, to be themost potent individual inhibitor of cancer ceils in in vitroexperiments. The cell viability was tested using the MTS in vitrocytotoxicity assay. This is considered the most convenient assay becauseof its ease of use, accuracy and rapid indication of toxicity (Malich Get al., Toxicology 124(3): 179-92 (1997).

The results obtained show that at high concentrations (40 micrograms/ml)of each. genistein showed an inhibition at 1, 2, 3 and 6 days ofincubation with an IC50 of 32, 22, 15 and 18 micrograms/ml, comparedwith IC50 values of 6, 6.5 and 7 for Compound A for the same periodsrespectively. More importantly, Compound A inhibited the growth of MCF-7cells even at low concentrations, namely 2.5 micrograms/ml and as earlyas within 8 hours of incubation and at days 1 and 2. By contrast, otherisoflavonoids including genistein at concentrations (<10 μM) enhancerather than inhibit the growth of MCF-7 cancer cells.

IC50 values observed for other compounds of the invention against MCF-7cells were as follows:

Compound of structure: IC50 (μg/mL) (IV)  6-10 (V) 10-20 (VI) 3.2 (VII)about 28 (VIII) <8  

The compound of structure (VI) was also tested against PC3 and LNCapcells and the IC50 values observed were 6.2 and 7.0 μg/mL respectively.

EXAMPLE 9 Comparative Inhibitory and Proliferative Effects of Daidzeinand Genistein, Their Methylated Analogues and Metabolites with5-hydroxy-O-Dma (Compound A) on MCF7 Cells

In vitro cell tissue culture experiments with MCF7 breast cancer cellswhen incubated with 5-hydroxy-O-Dma (Compound A) showed significantinhibition as compared with genistein, daidzein or their methylatedprecursors, namely formononetin and biochanin A or their metabolites forconcentrations of 15-40 μg/ml. This variation was more significant whencells were incubated for 8 hours where it was demonstrated that5-hydroxy-O-Dma had an IC50 of 6 μg/ml as compared with that ofgenistein which had an IC50 of >40 μg/ml for the same period ofincubation. Subsequent incubations at 24 hours, 48 hours, 72hours and144 hours revealed that the IC50 value of 5-hydroxy-O-Dma remainedbasically unchanged: ie remained in the range of 4-7 μg/ml. This is incontrast to the IC50 values obtained for genistein after incubations for48 hours (IC50=38) and 144 hours(IC50-15 μg/ml).

For concentrations of less than or equal to 10 μM of 5-hydroxy-O-Dma andgenistein, no significant inhibition was observed. However, in the caseof genistein, some proliferative activity of cancer cells wasdemonstrated at concentrations of less than or equal to 10 μM, whereas5-hydroxy-O-Dma showed no proliferative activity of cancer cells.

When daidzein, formononetin, biochanin A and other metabolites ofdaidzein and genistein such as dihydrodaidzein, tetrahydrodaidzein(transisomer). O-Dma, 6-hydroxy-O-Dma and equol were tested for theirinhibitory effect on MCF7 cells, it was found that with the exception ofbiochanin A and 6-hydroxy-O-Dma which showed some inhibition with anIC50 of 18-23 μg/ml at 72 and 144 hours incubation, all othermetabolites had no significant effect, with their IC50 values at about36->50 μg/ml.

These results suggest that compound A is a potent inhibitor of breastcancer cells but more importantly, compound A showed no proliferativeactivity of cancer cells at low concentrations as genistein docs. The6,7-dihydroxy groups in compounds of the invention appear to be criticalfor this difference of biological activity of compounds of the inventionwhen compared with analogues such as O-Dma and 6-hydroxy-O-Dma.

EXAMPLE 10 Comparative Inhibitory Effects of Daidzein and Genistein,Their Methylated Analogues and Metabolites with 5-hydroxy-O-Dma(Compound A) on Breast Cancer Cells

5-Hydroxy-O-Dma when tested with MDA-MB-468 (estrogen negative) cancercells showed significant inhibition at day 6 (IC50=6.8 μg/ml) ascompared with 8.8 μg/ml for genistein and 3-7 times more inhibitive whencompared with analogues of daidzein and genistein namely O-Dma (20μg/ml) and 6-hydroxy-O-Dma (43 μg/ml) respectively. The IC50 of5-hydroxy-O-Dma using MCF-7 estrogen positive breast cancer cells on day6 of incubation was 2.1 μg/ml for 5-hydroxy-O-Dma as compared with theanalogues of daidzein and genistein namely O-Dma (38 μg/ml) and6-hydroxy-O-Dma (33 μg/ml) respectively.

These results suggest that inhibition of 5-hydroxy-O-Dma like that ofgenistein, was more severe for the estrogen negative (−ve) cancer thanthat of the estrogen positive (+ve) cancer cells which suggests that inboth these cases the mechanism of action is not related to the estrogenreceptors.

EXAMPLE 11 Inhibitory Effects of Factor-2 on Breast Cancer Cells

Factor 2 was obtained by complete demethylation of glycitein after 4days of incubation with BBr₃. Incomplete demethylation gave a mixture ofglycitein and Factor 2. Alternatively, following fermentation ofdaidzein and glycitein from clover to give Factor-2, selectiveextraction and/or precipitation of Factor 2 from the fermentation mediumcan be easily achieved.

Factor-2 when tested with MCF 7 estrogen positive breast cancer cellsand MDA-MB-468 (estrogen negative) breast cancer cells showedsignificant inhibition of both types of cancer cells. Inhibition ofMCF-7 cells using Factor 2 gave IC50 values (at day 6 of incubation) of12 μg/ml and for MDA-MB-468 cells, the IC50 value was 8 10 μg/ml.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications which fall within itsspirit and scope. The invention also includes all of the steps,features. compositions and compounds referred to or indicated in thisspecification, individually or collectively, and any and allcombinations of any two or more of said steps or features.

1. A compound of the formula I or formula II

in which A is selected from the group consisting of

one of R₁ and R₂ is selected from H, OH and OCH₃, and the other of R₁and R₂ is selected from OH and OCH₃; one of R₃ and R₄ is selected fromH, OH and OCH₃, and the other of R₃ and R₄ is selected from OH and OCH₃;provided that at least one of the pairs R₁, R₂ and R₃, R₄ are both OH;R₅ is selected from OH and OCH₃; and

denotes a single or double bond; or a pharmaceutically acceptable saltor prodrug thereof; with the proviso that (a) when A is

and R₃ and R₄ are both OH then R₂ is other than H; and (b) when A is

and R₃ and R₄ are both OH and R₂ is OCH₃, then R₁ is other than H orOCH₃.
 2. A compound according to claim 1, wherein one of R₁ and R₂ isselected from H and OH, and the other of R₁ and R₂ is OH; one of R₃ andR₄ is selected from H and OH, and the other of R₃ and R₄ is OH; providedthat at least one of the pairs R₁, R₂ and R₃, R₄ are both OH; R₅ is OH;and

denotes a single or double bond.
 3. A compound according to claim 1 ofthe formula (IA) or (IIA)

wherein A is as defined in claim 1; R₂ is H, and R₁ is selected from OHand OCH₃; R₃ and R₄ are each OH; R₅ is selected from OH and OCH₃; and

denotes a single or double bond.
 4. A compound according to claim 1 ofthe formula (IB) or (IIB)

wherein A is as defined in claim 1; R₁ and R₂ are each OH; R₃ is H, andR₄ is selected from OH and OCH₃; R₅ is selected from OH and OCH₃; and

denotes a single or double bond.
 5. A compound according to claim 1which is 5-hydroxy-O-demethylangolesin (5-hydroxy-O-Dma)[1-(2,4,5-trihydroxyphenyl)-2-(4-hydroxyphenyl)-propan-1-one] having thestructure (IV):

or a pharmaceutical acceptable salt or prodrug thereof.
 6. A compoundaccording to claim 1 which is 3′-hydroxy-O-demethylangolesin(3′-hydroxy-O-Dma) [1-(2,4,dihydroxyphenyl)-2-(3,4-dihydroxyphenyl)-propan-1-one] having thestructure (V):

or a pharmaceutically acceptable salt or prodrug thereof.
 7. A compoundaccording to claim 1 which is3′-hydroxy-O-demethyldehydroangolesin(3′-hydroxydehydro-O-Dma)[1-(2,4-dihydroxyphenyl)-2-(3,4-dihydroxyphenyl)-prop-2-en-1-one] havingthe structure (VI):

or a pharmaceutically acceptable salt or prodrug thereof.
 8. A compoundaccording to claim 1 which is 3′-hydroxy-dihydrodaidzein having thestructure (VII):

or a pharmaceutically acceptable salt or prodrug thereof.
 9. A compoundaccording to claim 1 which is1-(2,4,5-trihydroxyphenyl)-2-(4-hydroxyphenyl)-prop-2-en-1-one havingthe structure (VIII):

or a pharmaceutically acceptable salt or prodrug thereof.
 10. Apharmaceutical composition comprising one or more compounds according toclaim 1, in association with one or more pharmaceutically acceptablecarriers, adjuvants, diluents and/or excipients.
 11. A food or drinkcomposition, which contains one or more compounds according to claim 1.12. A method for the treatment, prophylaxis, amelioration, defenseagainst, and/or prevention of a condition selected from the groupconsisting of menopausal syndrome; osteoporosis; rheumatic diseases;atherosclerosis; premenstrual syndrome; coronary artery spasm; vasculardiseases; Buergers Disease; migraine headaches; hypertension; benignprostatic hypertrophy; cancer; Alzheimers disease; inflammatorydiseases; baldness; psoriasis; acne; and diseases associated withoxidant stress; in a patient in need of said treatment, prophylaxis,amelioration, defense against, and/or prevention, which method comprisesadministering to said patient a therapeutically effective amount of oneor more compounds according to claim 1, either alone or in associationwith one or more pharmaceutically acceptable carriers, diluents,adjuvants and/or excipients.
 13. A method for the treatment,prophylaxis, amelioration, defense against, and/or prevention of ahormone-dependent condition in a patient in need of said treatment,prophylaxis, amelioration, defense against, and/or prevention,comprising administering to said patient a therapeutically effectiveamount of one or more compounds according to claim 1, either alone or inassociation with one or more pharmaceutically acceptable carriers,diluents, adjuvants and/or excipients.
 14. A method for the treatment,prophylaxis, amelioration, defense against, and/or prevention of ahormone-dependent condition in a patient in need of said treatment,prophylaxis, amelioration, defense against, and/or prevention,comprising administering to said patient a therapeutically effectiveamount of one or more compounds of formula I or formula II

in which A is selected from the group consisting of

one of R₁ and R₂ is selected from H, OH and OCH₃, and the other of R₁and R₂ is selected from OH and OCH₃; one of R₃ and R₄ is selected fromH, OH and OCH₃, and the other of R₃ and R₄ is selected from OH and OCH₃;provided that at least one of the pairs R₁, R₂ and R₃, R₄ are both OH;R₅ is selected from OH and OCH₃; and

denotes a single or double bond; or a pharmaceutically acceptable saltor prodrug thereof; said compound or compounds being administered eitheralone or in association with one or more pharmaceutically acceptableearners, diluents, adjuvants and/or excipients.
 15. A method for thetreatment, prophylaxis, amelioration, defense against, and/or preventionof cancer in a patient in need of said treatment, prophylaxis,amelioration, defense against, and/or prevention, comprisingadministering to said patient a therapeutically effective amount of oneor more compounds of formula I or formula II

in which A is selected from the group consisting of

one of R₁ and R₂ is selected from H, OH and OCH₃, and the other of Riand R₂ is selected from OH and OCH₃; one of R₃ and R₄ is selected fromH, OH and OCH₃, and the other of R₃ and R₄ is selected from OH and OCH₃;provided that at least one of the pairs R₁, R₂ and R₃, R₄ are both OH;R₅ is selected from OH and OCH₃; and

denotes a single or double bond; or a pharmaceutically acceptable saltor prodrug thereof; said compound or compounds being administered eitheralone or in association with one or more pharmaceutically acceptablecarriers, diluents, adjuvants and/or excipients.
 16. The methodaccording to claim 13 wherein said hormone dependent condition isselected from the group consisting of hormone dependent cancers, hormonedependent cardiovascular disorders and hormone dependent menopausaldisorders.
 17. The use of one or more compounds according to claim 1 forthe manufacture of a medicament for the treatment, amelioration, defenseagainst, prophylaxis and/or prevention of one or more conditionsselected from the group consisting of menopausal syndrome; osteoporosis;rheumatic diseases; atherosclerosis; premenstrual syndrome; coronaryartery spasm; vascular diseases; Buergers Disease; migraine headaches;hypertension; benign prostatic hypertrophy; cancer; Alzheimers disease;inflammatory diseases; baldness; psoriasis; acne; and diseasesassociated with oxidant stress.
 18. Use of one or more compoundsaccording to claim 1 for the treatment, amelioration, defense against,prophylaxis and/or prevention of one or more conditions selected fromthe group consisting of menopausal syndrome; osteoporosis; rheumaticdiseases; atherosclerosis; premenstrual syndrome; coronary artery spasm;vascular diseases; Buergers Disease; migraine headaches; hypertension;benign prostatic hypertrophy; cancer; Alzheimers disease; inflammatorydiseases; baldness; psoriasis; acne; and diseases associated withoxidant stress.
 19. A microbial culture or a food or drink compositioncontaining at least one microbial strain which microbial strain iscapable of producing one or more compounds according to claim 1 fromdaidzein and/or glycitein.
 20. A method for the treatment, prophylaxis,amelioration, defense against, and/or prevention of a hormone-dependentcondition in a patient in need of said treatment, prophylaxis,amelioration, defense against, and/or prevention, comprisingadministering to said patient a therapeutically effective amount of6,7,4′-trihydroxyisoflavone, or a pharmaceutically acceptable salt orprodrug thereof, either alone or in association with one or morepharmaceutically acceptable carriers, diluents, adjuvants and/orexcipients.
 21. The use of 6,7,4′-trihydroxyisoflavone for themanufacture of a medicament for the treatment, prophylaxis,amelioration, defense against, and/or prevention of one or moreconditions selected from the group consisting of menopausal syndrome;osteoporosis; rheumatic diseases; atherosclerosis; premenstrualsyndrome; coronary artery spasm; vascular diseases; Buergers Disease;migraine headaches; hypertension; benign prostatic hypertrophy; cancer;Alzheimers disease; inflammatory diseases; baldness; psoriasis; acne;and diseases associated with oxidant stress.
 22. The use of6,7,4′-trihydroxyisoflavone for the manufacture of a medicament for thetreatment, prophylaxis, amelioration, defense against, and/or preventionof a hormone-dependent condition.
 23. The method according to claim 14wherein said hormone dependent condition is selected from the groupconsisting of hormone dependent cancers, hormone dependentcardiovascular disorders and hormone dependent menopausal disorders.